A protein bactericidal for Pseudomonas aeruginosa has been isolated by polyacrylamide gel electrophoresis of a 0.01N HCl extract of granules obtained from normal human PMNL. The protein has a molecular weight of 57,000, as estimated by electrophoresis in sodium dodecyl sulfate polyacrylamide gel, and is bactericidal at concentrations as low as 9nM. The activity of this protein against P. aeruginosa was unaffected by changes in hydrogen ion and salt concentration. Bactericidal activity was dependent on the temperature of incubation and maximum activity of the bactericidal protein was observed after less than 2 minutes of incubation at 37 degrees centigrade. Chronic granulomatous disease (CGD) polymorphonuclear leukocytes were shown to be capable of killing P. aeruginosa strains in vitro, suggesting that the bactericidal protein is of functional significance in an oxygen-independent antimicrobial mechanism of intact PMNL. A correlation between the bacteria killed by CGD PMNL and those killed by extracts from CGD and normal PMNL granules was established.